Activation of SARS-CoV-2 by trypsin-like proteases in the clinical specimens of patients with COVID-19

SARS-CoV-2 enters host cells through the angiotensin converting enzyme 2 (ACE2) receptor and/or transmembrane protease, serine 2 (TMPRSS2). In this study, we investigated whether proteases increased SARS-CoV-2 infectivity using pseudotyped viruses and clinical specimens from patients with COVID-19. First, we investigated how trypsin increased infectivity using the pseudotyped virus. Our findings revealed that trypsin increased infectivity after the virus was adsorbed on the cells, but no increase in infectivity was observed when the virus was treated with trypsin. We examined the effect of trypsin on SARS-CoV-2 infection in clinical specimens and found that the infectivity of the SARS-CoV-2 delta variant increased 36,000-fold after trypsin treatment. By contrast, the infectivity of SARS-CoV-2 omicron variant increased to less than 20-fold in the clinical specimens. Finally, using five clinical specimens containing delta variants, enhancement of viral infectivity was evaluated in the presence of the culture supernatant of several anaerobic bacteria. As a result, viral infectivities of all the clinical specimens containing culture supernatants of Fusobacterium necrophorum were significantly increased from several- to tenfold. Because SARS-CoV-2 infectivity increases in the oral cavity, which may contain anaerobic bacteria, keeping the oral cavities clean may help prevent SARS-CoV-2 infection.

Real-time PCR. VeroE6 cells seeded in 96-well culture plates were treated with DMEM containing 10% FBS on ice for 10 min. Approximately 5 μL centrifugal supernatant of nasal and nasopharyngeal swabs, which were positive for SARS-CoV-2 in RT-qPCR, was added to the cells. After adsorption on ice for 60 min was complete, supernatants including the swabs were removed, and the cells were treated for 5 min with the indicated concentrations of trypsin or bacterial culture supernatants in DMEM containing 2% FBS that was prewarmed at room temperature. Then trypsin or bacterial culture supernatant was removed, and cells were cultured in DMEM containing 10% FBS at 37 °C for 24 h. RT-qPCR was performed to estimate the amounts of RNA. For RT-qPCR testing, we used the QIAamp Viral RNA Mini Kit (QIAGEN, Cat. No. 52904, Germantown, MD), which targets the N gene of SARS-CoV-2, and the assays were performed using QuantStudio 5 (Thermo Fisher Scientific Inc. Waltham, MA) 10 . RT-qPCR analysis was performed under the following conditions (reverse transcription: 50 °C for 5 min, 95 °C for 20 s; PCR: 95 °C for 75 s and 45 cycles of 95 °C for 15 s, 60 °C for 60 s). To quantify virus titer produced in the cells, we collected the cell culture supernatants, and determined the amount of RNA diluted tenfold using RT-qPCR.

Trypsinization experiments for SARS-CoV-2pv.
To examine the effect of trypsin after viral infection, VeroE6 cells seeded in 96-well culture plates were treated with DMEM containing 10% FBS on ice for 10 min. Approximately 100 μL of SARS-CoV-2pv (Wuhan, alpha, delta, and omicron) or VSVpv were added to the cells. After viral adsorption on ice for 60 min, the virus was removed, and the infected cells were treated for 5 min with various concentrations of trypsin (Sigma, T1426) in DMEM that was prewarmed at room temperature. After trypsin solution was removed, cells were cultured in DMEM containing 10% FBS at 37 °C for 24 h.
To examine the effect of trypsin on viral particles, SARS-CoV-2pv or VSVpv was exposed to various concentrations of trypsin in DMEM for 5 min at 37 °C. To inactivate trypsin, FBS was added at a final concentration of 20%. Then 20 μL of sample was added to each well, and the cells were incubated at 37 °C for 24 h. The infectivity of both SARS-CoV-2pv and VSVpv was assessed separately by measuring luciferase activity. The relative light unit value of luciferase was determined using the PicaGene Luminescence Kit (TOYO B-Net Co., Ltd., Tokyo, Japan) and GloMax Navigator System G2000 (Promega Corporation, Madison, WI), according to the manufacturer's protocol.

Syncytium formation.
To examine syncytium formation using S protein of SARS-CoV-2 variants after trypsin treatment, VeroE6 or 293 T cells seeded in 24-well culture plates were transfected with 0.5 μg of the indicated S protein of SARS-CoV-2-or VSVG-expressing plasmid together with 0.5 μg of pCAGGS-eGFP. At 48 h post-transfection, the cells were treated for 5 min with 800 µg/mL trypsin in DMEM containing 2% FBS that was prewarmed at room temperature. The trypsin-containing media was replaced with DMEM containing 10% FBS and incubated for 24 h. The cell monolayers were then observed for syncytium formation under a fluorescence microscope.
Addition of anaerobic bacterial culture supernatant to clinical specimens. We examined whether the culture supernatant of oral anaerobic bacteria increased SARS-CoV-2 infectivity. Three species of oral anaerobic bacteria: P. melaninogenica JCM6325, P. intermedia JCM11150, and F. necrophorum JCM3718 were obtained from Japan Collection of Microorganisms (RIKEN BRC, Ibaraki, Japan) and cultured using Brain Heart Infusion medium (Becton, Dickinson and Company, Cat www.nature.com/scientificreports/ were added to VeroE6 cells that had been chilled on ice for 10 min. Incubation was performed for 60 min on ice for the virus to adsorb on the cells. Then the virus was removed, and the cells were treated with the culture supernatant of each anaerobic bacteria at 37 °C for 5 min. Then the culture supernatant was removed, and the cells were cultured in DMEM containing 10% FBS at 37 °C for 24 h. To measure the viral titer produced in the cells, the cell culture supernatants were collected, and the amount of RNA diluted tenfold was determined by RT-qPCR.

Statistical analysis. The statistical significances were analyzed by the Dunnett's test, the Mann-Whitney
U-test, and the Student's t-test for experiments using SARS-CoV-2pv, using clinical specimens with trypsin treatment, using clinical specimens with bacterial culture supernatants, respectively. P values < 0.05 were considered to be significant. All statistical analyses were performed using IBM SPSS Statistics (v.27; IBM Corp., Armonk, NY, USA).
Ethical approval. This study was performed in accordance with the Helsinki Declaration and was approved by the ethical review board of the Toyama Institute of Health (approval No.: R2-12). The need to obtain written informed consent was also waived by the ethical review board of the Toyama Institute of Health because of the anonymous nature of the data. Instead, we announced the study officially and ensured that patients could opt of the study.

Results
Trypsin treatment increases SARS-CoV-2pv infectivity. SARS-CoV-2 is highly regulated legally and must be handled in facilities with biosafety level (BSL) 3. Therefore, the Wuhan, alpha, delta, and omicron variants of SARS-CoV-2pv were generated using the vesicular stomatitis virus (VSV) pseudotyping system. Using these viruses allowed us to perform experiments in a BSL2 laboratory. In addition, because luciferase was inserted as a reporter gene, the efficiency of infection was quantitatively evaluated using luciferase activity as an index. Furthermore, the use of pseudotyped viruses eliminates the effects of components derived from clinical specimens.
Trypsin increased the infectivity of the Wuhan, alpha, and delta variants of SARS-CoV-2pv in a concentrationdependent manner-up to ~ tenfold at 800 μg/mL (Fig. 1A). By contrast, trypsin treatment did not increase the infectivity of the SARS-CoV-2pv omicron variant.
Next, we examined the action of trypsin treatment temporally and treated SARS-CoV-2pv with trypsin before infecting cells. We found no increase in infectivity effect in any variant (Fig. 1B). These results suggest that trypsin increases SARS-CoV-2pv infectivity after the virus is adsorbed on the cell and does not activate viral particles. In this study, the concentration of trypsin treatment was determined with reference to a previous report on effects of proteases for SARS-CoV infection by Matsuyama et al. 6 . Although the concentration is much higher than the concentration of general treatment, the treatment time is as short as 5 min. Therefore, even under this condition, it is considered that there is little effect on cell toxicity.

Syncytium formation of S protein-expressing cells after trypsin treatment. To investigate
whether the fusion of S protein of SARS-CoV-2 and host cellular membrane is involved in the trypsin-mediated increase in infectivity, we compared syncytium formation in cells expressing S protein after trypsin treatment (Fig. 2). VeroE6 cells expressing S protein of the Wuhan, alpha, and delta variants of SARS-CoV-2 formed particularly large syncytia after trypsin treatment ( Fig. 2A). This phenomenon was confirmed in 293 T cells expressing S protein of the SARS-CoV-2 delta variant, although cells expressing S protein of SARS-CoV-2 Wuhan and alpha variants exhibited weak fusion activity (Fig. 2B). Almost no syncytium formation was observed in cells expressing S protein of the SARS-CoV-2 omicron variant or VSVG in both cell types. These results indicate that the omicron variant shows weaker fusion activity than other variants after trypsin treatment.

Trypsin treatment increases SARS-CoV-2 infection in clinical specimens.
First, we examined the effect of trypsin treatment on SARS-CoV-2 infection in clinical specimens. Clinical specimens were used after centrifugation at 10,000×g for 30 min to avoid bacterial contamination in cell culture. Almost no bacterial contamination has been observed in our viral isolation methods as described previously 11 . In 56 clinical specimens containing the SARS-CoV-2 delta variant, trypsin treatment was found to increase the infectivity by 4.2-to 36,000-fold in all compared with untreated specimens (Fig. 3A). By contrast, the infectivity of 33 specimens among the 44 SARS-CoV-2 omicron variants increased after trypsin treatment by 0.3-to 18.4-fold, except one specimen that exhibited 24,000-fold higher infectivity than the specimen not treated with trypsin (Fig. 3B). Eleven specimens had equal or low levels of infectivities compared with the specimen not treated with trypsin. Next, the significance of the increase in the infection of delta variant and omicron variant with trypsin treatment was statistically analyzed using boxplots (Fig. 3C). As a result, trypsin-induced infection was significantly enhanced in clinical specimens containing delta variants. Thus, trypsin treatment increased the infectivity of the omicron variant to a lower extent than that of the delta variant. We observed no significant difference in the Ct values of specimens with or without increased infectivity. We speculate that the effect of trypsin treatment on increasing infectivity is not simply affected by viral load.

Enhancement of SARS-CoV-2 infection by culture supernatants of anaerobic bacteria.
We examined whether culture supernatants of anaerobic bacteria have the same effect as trypsin on enhancing infection ( Fig. 4A-C) www.nature.com/scientificreports/ enough to be used for further experiments, were used to determine whether culture supernatants of oral anaerobic bacteria (Prevotella melaninogenica, Prevotella intermedia, and Fusobacterium necrophorum) increased infectivity. Treatment with culture supernatants of P. melaninogenica or P. intermedia increased infectivity negligibly. By contrast, treatment with culture supernatant of F. necrophorum significantly enhanced viral infection by ~ tenfold compared to treatment with medium alone in all the specimens examined in this experiment.

Discussion
Trypsin treatment of cells is known to increase viral infectivity and to promote membrane fusion in animal and human coronaviruses, including SARS-CoV 6,12,13 . SARS-CoV-2 virus entry was also reported to increase in the presence of trypsin 3,14 . Our findings revealed that trypsin treatment of cells increased SARS-CoV-2pv infectivity as well as infectivity of SARS-CoV-2 present in clinical specimens derived from the nasal and oral cavities of patients with COVID-19. The increase in infectivity was different among SARS-CoV-2 variants. In clinical specimens from patients with COVID-19, the delta variant showed up to tens of thousands-fold increase in infectivity after trypsin treatment, whereas the omicron variant showed only a few to several dozen-fold enhancement. We observed no significant difference in Ct values between clinical specimens in which infectivity increased greatly after trypsin treatment and those in which infectivity did not increase, suggesting that the increase in infectivity www.nature.com/scientificreports/ after trypsin treatment is not simply affected by viral load. Although this difference is thought to be caused by differences in the components contained in the clinical specimens, the detailed reason is unknown and further investigation will be needed. Studies have shown that infection by members of the Orthomyxoviridae and Paramyxoviridae families, including the influenza virus, are facilitated by some proteases [15][16][17][18] . Because the envelope glycoproteins of these viruses are not completely cleaved in de novo synthesized cells, the viral particles produced from these cells contain partially-cleaved or uncleaved glycoproteins. Treatment with proteases cleaves the glycoproteins on the virions, thus enhancing their infectivity. Thus, trypsin acts directly on virions to increase infectivity. However, the infectivity of SARS-CoV was reported to increased when S protein is cleaved by proteases after binding to the cellular receptor(s) such as ACE2 6 . In SARS-CoV-2 infection, the increase in infectivity after treatment with trypsin or other proteases may be due to a mechanism different from that for influenza virus or other viruses. Each SARS-CoV-2pv variant was pretreated with trypsin before being used to infect the cells; however, this treatment did not increase the infectivity of SARS-CoV-2pv. In other words, the protease can induce the fusion activity of S protein only after the virus binds to the cellular receptor. Studies have shown that the omicron variant uses a TMPRSS2-independent entry pathway 19,20 . Surprisingly, fusion activity by trypsin treatment was observed in S protein-expressing VeroE6 and 293 T cells, which slightly and hardly express ACE2, respectively. This suggests that the enhancement of infection by trypsin treatment is not necessarily ACE2-dependent, but may be induced if it can bind to cells, especially for the S protein of delta variant. The results that fusion activity tended to be stronger in the delta variant S protein-expressing cells and weaker in the omicron variant S protein-expressing cells are consistent with infectivity of the virus with trypsin treatment. In SARS-CoV-2 infection, syncytium formation in infected cells has been reported to play a role in viral replication and pathogenesis of severe COVID-19 21,22 . Moreover, cellular membrane fusion activity of the omicron variant was reported to be lower than that of the delta variant 19 . Studies have reported that cell membrane fusion is activated by treatment with buffers with an acidic pH 23 . In this study, we newly found that trypsin-like proteases activate cell membrane fusion.
Furthermore, we found that the infectivity of SARS-CoV-2 from the nasal cavity increased in the presence of culture supernatants of oral anaerobic bacteria, such as F. necrophorum. It is unclear why SARS-CoV-2 infectivity increased only when culture supernatants of F. necrophorum were used, but not when P. melaninogenica and P. intermedia were used. Therefore, the amount and type of proteases in the culture supernatant must be evaluated and currently under consideration. These are a type of commensal bacteria that are present in the oral cavities of healthy people and have been reported to be involved in the exacerbation of periodontal disease and gingivitis 24,25 . Studies have shown differences in the nasopharyngeal microbiome of patients with COVID-19 26 . In addition, although operational taxonomic units classified as Prevotella were found to be significantly more The eGFP-expressing plasmid was co-transfected for visualization. Syncytium formation was imaged using a fluorescence microscope.  www.nature.com/scientificreports/ abundant in patients who developed more severe COVID-19, the mechanism of interaction between the salivary microbiome and SARS-CoV-2 remains to be elucidated 27 .
In summary, our findings revealed that SARS-CoV-2 in the nasal or oral cavity of patients with COVID-19 is activated by the action of proteases. Therefore, keeping the oral cavity clean on a daily basis and reducing components derived from oral indigenous bacteria will help prevent SARS-CoV-2 infection. Furthermore, it is better to think positively about oral hygiene, including chemical plaque control in patients at a higher risk of severe COVID-19.

Data availability
The data used to support the findings of this study are available from the corresponding author upon request.  The results shown are from three independent assays, with error bars representing standard deviations. Significance was determined by the Student's t-test in comparison to the data of no treatment (Ctrl): *P < 0.05; **P < 0.01.